title: β-Catenin knockdown in liver tumor cells by a cell permeable gamma guanidine-based peptide nucleic acid.
abstract: Hepatocellular cancer (HCC) is the third cause of death by cancer worldwide. In the current study we target β- catenin, an oncogene mutated and constitutively active in 20-30% of HCCs, via a novel, cell permeable gamma guanidine-based peptide nucleic acid (γGPNA) antisense oligonucleotide designed against either the transcription or the translation start site of the human β-catenin gene. Using TOPflash, a luciferase reporter assay, we show that γGPNA targeting the transcription start site showed more robust activity against β-catenin activity in liver tumor cells that harbor β-catenin gene mutations (HepG2 & Snu-449). We identified concomitant suppression of β-catenin expression and of various Wnt targets including glutamine synthetase (GS) and cyclin-D1. Concurrently, γGPNA treatment reduced proliferation, survival and viability of HCC cells. Intriguingly, an angiogenesis quantitative Real-Time-PCR array identified decreased expression of several pro-angiogenic secreted factors such as EphrinA1, FGF-2, and VEGF-A upon β-catenin inhibition in liver tumor cells. Conversely, transfection of stabilized-β-catenin mutants enhanced the expression of angiogenic factors like VEGF-A. Conditioned media from HepG2 cells treated with β-catenin but not the mismatch γGPNA significantly diminished spheroid and tubule formation by SK-Hep1 cells, an HCC-associated endothelial cell line. Thus, we report a novel class of cell permeable and efficacious γGPNAs that effectively targets β-catenin, a known oncogene in the liver. Our study also identifies a novel role of β-catenin in liver tumor angiogenesis through paracrine mechanisms in addition to its roles in proliferation, survival, metabolism and cancer stem cell biology, thus further strengthening its effectiveness as a therapeutic target in HCC.
authors.affiliation: Endowed Chair for Experimental Pathology, Director- Division of Experimental Pathology (EP), Professor of Pathology (EP) & Medicine (GI, Hepatology and Nutrition), University of Pittsburgh School of Medicine, 200 Lothrop Street S-422 BST, Pittsburgh, PA 15261, USA. smonga@pitt.edu.
authors.forename: Evan
authors.initials: E
authors.lastname: Delgado
authors.forename: Raman
authors.initials: R
authors.lastname: Bahal
authors.forename: Jing
authors.initials: J
authors.lastname: Yang
authors.forename: Jung M
authors.initials: JM
authors.lastname: Lee
authors.forename: Danith H
authors.initials: DH
authors.lastname: Ly
authors.forename: Satdarshan P S
authors.initials: SP
authors.lastname: Monga
mesh_terms: D042501:Angiogenic Proteins / Q000235:genetics / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D049109:Cell Proliferation / Q000187:drug effects; D057846:Cell-Penetrating Peptides / Q000737:chemistry / Q000494:pharmacology*; D055785:Gene Knockdown Techniques; D019791:Guanidine / Q000737:chemistry*; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D008156:Luciferases / Q000378:metabolism; D009389:Neovascularization, Pathologic; D020135:Peptide Nucleic Acids / Q000737:chemistry / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D024363:Transcription Initiation Site; D014407:Tumor Cells, Cultured; D051176:beta Catenin / Q000037:antagonists & inhibitors* / Q000235:genetics
chemical_list: D042501:Angiogenic Proteins; C495270:CTNNB1 protein, human; D057846:Cell-Penetrating Peptides; D020135:Peptide Nucleic Acids; D012333:RNA, Messenger; D051176:beta Catenin; D008156:Luciferases; D019791:Guanidine